COVID-19 Waves with Associated Variants & Scenario in Pakistan

COVID-19 in Pakistan

World Health Organization declared COVID-19 as an emergency on 30th January 2020 and a pandemic on 11th March [14]. In Pakistan, it was reported firstly on 26th February 2020 by the Pakistan government when two persons were found infected with the disease. Both patients returned from Iran and were declared positive for the disease in big cities of Pakistan, Islamabad, and Karachi [15]. Lahore General Hospital was the first public health institute that immediately started COVID-19 diagnosis and SARS-CoV-2 detection through real-time PCR [16].

Two deaths caused by COVID-19 were observed in Khyber-Pakhtunkhwa on 18th March 2020 [15]. It was observed that 98% of Pakistani patients recovered while the mortality rate was 2% [17].

Pakistani people including healthcare professionals and the public were trained through webinars, seminars, conferences, and other awareness campaigns to fight against this pandemic. Still, this disease created depression and anxiety among people including medical and para- medical staff due to the overload of work, financial and appropriate facility crises. In a survey study, it was observed that 92.6% were familiar with COVID-19 disease, while the prevalence of depression and anxiety was 14.3% and 4.6%, respectively. Healthcare professionals and medical students were observed to have less depression and anxiety when compared with the general public [18].

Common symptoms in Pakistani patients at the onset of Covid-19 illness were low-grade fever, malaise, cough, chest pain, sore throat, tightness, and shortness of respiratory rate. As SARS-CoV-2 causes respiratory problems therefore computed tomography (CT) scans and radiological examinations also prove helpful for diagnosis [19]. In a study, high-resolution CT scans and Real-Time PCR tests were compared to check the sensitivity for Covid-19 diagnosis, and noted that HRCT has higher sensitivity (92%) than RT-PCR (45%) [20]. But as we know the respiratory complications and symptoms of COVID-19 are also associated with a number of other illnesses therefore molecular especially nucleic acid-based detection of coronavirus is important in COVID-19 diagnosis [21].

Severe Acute Coronavirus-2 and Mutations in its Genome

SARS-CoV-2 is coronaviridae-related beta-coronavirus. The virus is spherical in shape having spikes on the surface, enveloped, and its genome (RNA) can be directly translated into proteins due to its 5’-3’ orientation. Non-structural- protein 14 (NSP14) possesses proofreading capability thus keeping the mutation rate slow. Any change in nucleotide sequence is known as mutation and the genomes that possess different genetic sequences are called mutants or variants. There are two ways through which virus change their genomes constantly, antigenic shift and antigenic drift. With antigenic drift, SARS-CoV-2 mutates itself and gets rid of the host immune system’s recognition. The COVID-19 variants are classified as variants of concern, variants of interest, and variants of high consequences by Center of Disease Control and Prevention. Variants along with increased spread, illness, and mortality, also escape detection tests and hence pose new challenges in diagnosis and treatment [22]. According to the WHO coronavirus dashboard, 612.72 million COVID-19 cases and 6.51 death reported as of 25th September 2022 [23].

Out of four genera of coronaviruses the alpha and beta genera viruses possess the ability to cause illness in humans. Zoonotic SARS-CoV-2 is very similar to bat coronaviruses [24] RmYN02 and RaTG13 with 93.3% and 96.2% genetic homology, respectively [25, 26]. Malayan pangolin coronavirus also found genetic similarity with SARC CoV-2 [27, 28, 29, 30, 31].

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Every continent of the world has been affected due to COVID-19. The global health crisis was caused by the emergence of SARS-CoV-2 strains [28]. A number of its variants evolved, and everyone posed destruction in their own way of severity, transmission, drug resistance, and mortality rate. All are associated with decreased immune response from previous infection or immunization resulting in subsequent pandemic outbreaks [29, 30]. Different studies reported various mutated genes, E, S, M, N, ORF3a, ORF1ab, ORF6 to ORF10, and spike mutations associated with the emergence of new SARS-CoV-2 variants i.e., alpha, beta, gamma, delta [28], and Omicron [31] as shown in Figure 1.

Waves of COVID-19 and Associated Variants in Pakistan

The mutation frequency of SARC-CoV-2 increased in the pandemic period from 12.19% to 23.63%, 31.03%, and 41.22% during the 1st wave to the 2nd, 3rd and 4th wave, respectively [33]. Omicron has 5-11 times the mutation frequency than previous variants [34]. The most common strains associated with 1st wave were B.1.160, B.1.255, and B.1.36, 2nd wave B.1.247 and B.1.36, 3rd wave B.1.1.7 and B.1.36, and 4th wave B.1.617.2 (Delta strain) [33]. It was noted that B.1.36 strain was observed during all four waves of COVID-19 which shows their fitness survival and drug & vaccine resistance capability. The 5th wave of this disease was due to the novel variant Omicron B.1.1.529 [35, 36], while the new variant BA.5 of omicron is known to be responsible for the sixth wave in Pakistan [37]. The 5th VOC Omicron (B.1.1.529) originated in Botswana, Africa in November 2021 and surprised the other VOCs with its global dominance [38] and antibody circumvention [39].

Six COVID-19 waves in succession have been reported in Pakistan; the 1st from March to July 2020, 2nd October 2020 to January 2021, 3rd from April to May 2021 [40], 4th from July to September 2021, 5th from December 2021 to May 2022 [36, 41], and the sixth wave from June 22 to present [37].

Corona’s first wave in Pakistan

Duration: February to July 2020: The first wave of COVID-19 was very uncertain because of the unknown virus nature, pathogenicity, infectivity mechanism, transmission, and treatment options. Pakistan and other countries managed somehow to control the mortality rate because of rapid response and strategies. The initial level of transmission control is very necessary for any kind of outbreak to lower the fatality. Pakistani govt put partial, smart lockdowns, and designed new policies and SOPs for the movement of people from one place to another. The first wave in Pakistan lasted for 7 months from February to September 2020 [42].

The Wuhan strain originated in January 2020 and was of L clade (a viral clade is a group of viruses that share the same genetic changes and belongs to a common ancestor) isolate that later diversified itself into several other clades and subclades such as G, S, and V [43]. During the first wave, S and G clade strains were observed in Pakistan [44]. Initially out of 6 noncoding and 8 coding genes, ORF1B and nucleocapsid genes were observed with great variation [45]. Then by the end of 2020 spike glycoprotein genes mutated at a very higher frequency and give rise to a number of variants of concern all around the world [46]. Further mutations in S and N genes, ORFIAB and ORF3A classified strains into G, I, L, S, V, GH, GR, GV, and subclades by GISAID [47, 48].

The research of Pakistani SARC-CoV-2 genomic sequences that were originated during 2020 and 2021 and saved in the PANGOLIN database showed that 58% of sequences from April-June 2020 were of B.1. Along with B.1 the next lineage was A, B.1.1.1 and B.1.36. Whereas, lineage B.1.1.1 and A were studied in sample sequences before August, and B.1.36 and B.1 were seen after August 2020 [49].

In Pakistan first wave was short with a low mortality rate in which timely management policy has a significant role. A large number of infected people, active cases, deaths, and recoveries were observed upon the end of the first wave [36, 50] as shown in Figure 1 & Figure 2.

The highest mutation incidence recognized during the first wave was in non-structural protein 3 and spike protein. Particularly, the spike protein showed the highest rate of homoplastic sites which includes the D614G mutation, missense SNVs, and Q677H mutation. Among the other frequently repeated variations are 241:C>T, a mutation in the upstream 5′-UTR, the nsp3 silent mutation F924F, missense mutation P4715L in nsp12, silent mutation L227L in nsp13, silent mutations D294D, silent mutation L280L in nsp14, G880G mutation in spike protein, silent mutation Y71Y, missense mutation Q57H in orf3a, and H125Y membrane missense mutation, E39X stop-gain mutation of orf7b, and R209I and S194L nucleocapsid missense mutations [33].

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Corona second wave in Pakistan

Duration: October 2020 to February 2021: To avoid increased treatment and vaccination SARC-CoV learned to resist them and invade the immune system [42]. A second wave of Covid-19 was raised on 28th October 2020 and lasted till February 2021 in Pakistan. This wave infected 579,973 people, with 22,098 active cases, 12,860 mortalities, and 546,371 recoveries [36, 50] as shown in Figure 2 & Figure 3. This wave occurred due to B.1.351/20H/501Y.V2 variant of South Africa. Genetic studies of the virus identified different variants of lineage B.1 and B.6 in Pakistan during this period [52].

During the second wave of COVID-19, Rehman et al., 2022 observed B1.562, B1.36.31, and lineages in Pakistan from the PANGOLIN database. It was also observed that from December 2020 to January the sequences were of alpha lineage B.1.1.7. While in Bangladesh beta variant was responsible for COVID-19 second wave.

The B.1.351/501Y.V2 was recognized firstly in October 2020 in Nelson Mandela Bay of South Africa and then in December 2020 in Zambia. The 17 amino-acid changes were found in the S protein with 23 mutations in which notable mutations were N501Y, K417N, and E484K. Antibody escape was performed mainly by the E484K mutation associated with this variant, the main reason for reduced vaccine sensitivity [53]. Studies have shown lower efficacy of Janssen, Novavax, and Astra-Zeneca vaccines against this variant [54].

B.1.1.250 lineage was associated with Covid patients during May and late 2020 in Bangladesh, the United Kingdom, the USA, Australia, and Europe. While lineage B.1.261 was recognized in Saudi Arabia and then disseminated to Pakistan. Other VOC that emerged during the second wave includes UK variant B.1.1.7 [55], South Africa variant B.1.3.51 [56], and P.1 [57] (Japan. 2021) with an increased rate of transmissibility from 50 to 70% [52].

Variants during the COVID wave such as B.1.36.6 and B.1.36 lineages were closely related to subclades from India and the United Arab Emirates. B.1.247 strain was in close association with Iranian sub-clades, B.1.160 strain with India, and B.1.36 with India and Iran. B.1.1 genomes of SARC- CoV-2 during the second wave in Pakistani patients were in close peroxidation with sub-clades from Iran, India, UAE, and Israel [33].

Corona third wave in Pakistan

Duration: Late March to May 2021 [40]: From late March to May 2021, Pakistan experienced the third wave [58]. By the PANGOLIN database [55] occurrence of B1.1.7 lineage served as confirmation for the beginning of the third wave and its peak following March. Other B.1.1.7 sub-lineages, like Q.4 and Q.1, were first noticed starting in April 2021 although they haven’t consistently been shown in a lot of sequences during the following months. It is interesting to note that none of the sequences examined in the months following April 2021 contained the B.1.36.31 lineage. The Beta strain B.1.351 was detected in Pakistan for the first time in March 2021, and most cases were recorded in May and June of that same year. Meanwhile, the lineage B1.617.2 (Delta) initially appeared in April 2021, and since then, its numbers have been continuously rising by greater than 100 percent in the month of August 2021 relative to the other sequences observed in July 2021 and onward. The three main lineages identified during the third wave that continued from May to July 2021 were B.1.1.7, B.1.617.2, and B.1.351 [49].

Since about January 27, 2021, more than 64 nations, including Pakistan  [59] had been found to have the novel SARS-CoV-2 strain known as 20I/501Y.V1 and B.1.1.7, which originated in the United Kingdom. B.1.1.7 was associated with a greater death rate in Pakistan as compared to other variants of this wave. The ten major cities in Pakistan; Rawalpindi, Hyderabad, Faisalabad, Islamabad, Bahawalpur, Lahore, Swat, Multan, Muzaffarabad, and Peshawar, were placed under a rigorous lockdown till 11th April 2021, and the provincial administration was instructed to adhere with SOPs. [42] and the total cases, active cases, deaths, and recoveries till the end of this wave were 922,824, 57,336, 20,850, and 844,638 respectively as shown in Figure 1 & Figure 2.

The time of start, which was Spring, was common during the period of the first wave and third waves (from March to April). So, it could be hypothesized that the increased transmission of this SARS-CoV-2 was significantly based on the pollens during this period [42].

In September 2020, B.1.1.7 was found in the UK, and in contrast to the initial strain discovered in Wuhan, China, it includes 23 mutations. The S protein was found to contain eight of all these mutations. N501Y and P681H 69/70 deletion are significant mutations in this gene. The S protein seemed to be able to attach to the ACE2 receptor more firmly because of the N501Y mutation [60]. Compared to the other two, it was 40–80% more transmissible [61] and was responsible for almost 5,000 deaths out of 17,452 deaths due to COVID-19 between September and February. When compared to other variants, it was calculated that the mortality was about 55% greater [62]. Similarly to this, research reports from January 2021 also showed that this variant had a higher fatality rate [63]. 82 different countries have reported this variant [57].

Various research projects have been undertaken to evaluate the efficacy of anti-inflammatory and antiviral drugs in the management of the UK strain [22].

B.1.617.2 and B.1.351 were third-wave variants from Pakistan which are delta and beta variants respectively. As of June 20, 2021, the beta variant B.1.351, analyzed in over 95 nations including Pakistan, was known for having three major mutations at specific residue sites known as receptor-binding domain (RBD) in its spike, specifically N501Y, E484K, and K417N [64]. Furthermore, because of the E484K mutation in the spike protein, the South African variant showed an immunological escape strategy against antibodies [65]. This variant has properties to escape from monoclonal antibodies and serum antibody reactions [66].

B.1.617 variant was 1st time appeared in Maharashtra and then spread to the whole state along with some other countries. This variant has two sub-lineages of B.1.617.1 and B.1.617.2. From these, sub-lineage B.1.617.1 has P681R, E484Q, and L452R mutations whereas B.1.617.2 sub-lineage has P681R, T478K, and L452R mutations in its spike at receptor-binding domain. The mutations in the receptor- binding domain (RBD) boosted the infectivity because of the presence of T478K and L452R by increasing the attachment of its spike protein to the receptors of ACE2 in humans [67, 68]. Both mutations lessen the monoclonal antibodies’ ability to bind to specific molecules, which reduced their capacity to neutralize. Furthermore, structural examination of mutations in the cleavage site of furin, L452R, E484Q, and P681R demonstrated higher ACE2 interaction and cleavage rate, leading to greater transmissibility [69]. The majority of the patients affected with this variant were found in Islamabad (63%), followed by Azad Kashmir and Peshawar (13% each) as compared to the Beta (B.1.351) variant mostly found in Islamabad (56%) and then in Peshawar (22%), Azad Kashmir (11%) and Rawalpindi (11%).

In this wave, nsp12 & N genes were highly mutated after S and nsp3 genes with 28, 27, 39, and 35 mutational sites respectively. In addition to the D614G, other spike mutations like N501Y, p.68 70del, A570D, p.143 144del were reported in its genome. From these, N501Y and p.68 70del were the distinguishing features of the UK B.1.1.7 variant. These two genomes contained an E484K mutation like the B.1.351 South African variant [33].

The third wave’s genomes were predominantly made up of UK variant B.1.1.7 which accounted for sixty-nine percent of the total genomes along with B.1.36, B.1.351, B.1.468, B.1.1.413, and A.27 lineages genome. It was significant to note that there was no Alpha variation in the Quetta genomes and this city has minimal contact with the other provinces. In Karachi, Pakistan’s capital and largest city, the South African variant was seen. ML phylogeny was carried out by using SARS-CoV-2 genomic data from various countries to determine the possible mode of transmission of all these variants. In the case of France, Italy, and England this genomic data was grouped with the South African variant of Karachi. Some B.1.1.7 variants of Mardan and Lahore were also found in England and India whereas others were found in the United States, Italy, and England. These two findings show that Europe and India were the two main entry points for the pandemic’s third wave virus [33].

Corona fourth wave in Pakistan

Duration: July to September 2021: The fourth wave started in July 2021 in Pakistan [70]. The Delta variant first appeared in the summer of 2021 and quickly became a global sensation. It was in a worse state when it arrived in Pakistan. Luckily, a vaccine that was effective against the Delta form had been approved. Over 1,245,000 cases with 47,331 active cases, 22,781 deaths, and 919,163 recoveries at the end of this wave were observed as shown in Fig. 1 and Fig 2. Smart restrictions and lockdowns were implemented in key cities including Lahore, Islamabad, Rawalpindi, Muzaffarabad, Faisalabad, Mirpur, Multan, Peshawar, Abbottabad, Karachi, Gilgit, Hyderabad, and Skardu. To prevent the delta virus from spreading, workplaces and schools were shuttered. The construction of several vaccination facilities and the availability of a wide variety of vaccinations helped to advance the immunization process [36].

Brazilian variant (501Y.V3/P.1 or 20J)

The P.1 variant referred to as B.1.1.28.1, was discovered for the 1st time in Manaus, North Brazil in December 2020. It was discovered in samples examined at the National Institute of Infectious Diseases (NIID) of Japan in January 2021 in Brazilian travelers at the Haneda airport, just outside of Tokyo, during a standard passenger screening test. It seemed to have 35 mutations and 17 alterations to the amino acids. The mutations N501Y, E484K, and K417T were significant ones. According to research, this variant was nearly equally infectious in both older (greater than 60 years old) and younger (18 to 59 years old) individuals. It seemed to be 2.2 times more transmissible and resulted in few occurrences of reinfection after recovering from COVID-19 [71]. The vaccine’s effectiveness against the P.1 strain was anticipated to be similar to that of B.1.351 because both strains have identical receptor binding alterations. So, the  decreased effectiveness of the vaccine against the B.1.351 variant also resulted in decreased effectiveness against the P. 1 variant. Laboratory tests conducted by Sinovac Biotech have shown that the Corona vaccine has a 50 percent  success rate in eliminating P.1 variant acquisition in Brazil [72].

Double mutant variant (B.1.617)

It was India where this variant was discovered first. This variant was referred to as a “double mutant” because two mutations occur in this  variant. On April 5, 2021, its first case was reported in San Francisco, USA. L452R and E484Q were two significant mutations. These variants had a higher likelihood of spreading and were also immune to immunization.

According to the PANGOLIN database [55], B.1.617.2 and B.1.617.2 variants along with their sub-lineages were among the most common lineages during the fourth wave. These sub-lineages initially appeared in the month of May and then in August 2021. In December 2021, a new BA.1 lineage of Omicron was discovered in Pakistan. It was interesting to see that the isolates were having lineages A and B. A.1. [49].

During the fourth wave, the delta 21J (known as B.1.617.2) and 21I variants of the genome were discovered. The Delta variant was the most lethal and highly contagious with severe illness signs. As of June 24th, 2021, 77 countries have identified this variant [29, 73]. The Delta variety has had severe effects in the UK. More comprehensive molecular testing technologies and strict quarantine regulations were developed for international travelers to stop the rapid spread of this strain in the population. On September 15–20, 2021, GISAID CoV-Surver analysis of all the examined genomic samples revealed that they were all positive for Delta variants. The most prevalent mutations found in all genomic isolates were T478K, L452R, T19R, E156G, F157del, P681R, R158del, D614G, and G142D. The S protein’s RBD showed a T478K mutation that affected how it interacts with human ACE2 [74, 75]. The T478K, which was present in the epitope area of powerful neutralizing monoclonal antibodies, was specific to the Delta variant [76].

Delta variant has affected >100 countries. Humans were seriously harmed by the delta variant. Many people died as a result of this variant. Additionally, a lot of people experienced difficulties in getting medical care and dealing with oxygen cylinders. However, the delta version of the coronavirus proved particularly dangerous for humans. This virus caused a lot of losses. A very large death rate was associated with this wave [77].

South Africa’s 4th  wave was mostly caused by three Omicron variants (BA.3, BA.2, and BA.1). The most destructive Delta variant was succeeded by the BA.1 variant in late 2021, and it quickly rose to prominence. At the onset of  March 2022, the BA.2 variant of Omicron succeeded it, and by late April 2022, it had overtaken it as the most prevalent variant globally [78, 79].

Corona Fifth Wave in Pakistan

Duration: December 2021 to May 2022 [36, 41]: In the fifth wave of COVID-19, a new variant of Omicron (or B.1.1.529) was reported in Botswana, South Africa in November 2021. Fig. 2 and Fig. 3 show the total number of cases, active cases, deaths, and recoveries of patients from COVID-19 experienced during this wave. Omicron has destroyed every city in its path. Compared to the delta virus, Omicron was more (double) transmissible but not as deadly as earlier variants were. All ages, more men, both vaccinated and unvaccinated individuals were affected, and re-infection was observed [36]. This variation has undergone 50 mutations, while the spike protein has undergone more than 30 modifications [80].

This variant was reported in Israel, the UK, Australia, the Netherlands, Germany, Italy, Belgium, Hong Kong, and Botswana after its discovery in South Africa [81]. The human immune cells release antibodies that primarily affect its spike protein in order to treat COVID infection. This variant showed multiple mutations which played a great role in its escape from neutralization and the immune system. That’s why researchers were not clear about the effectiveness of its vaccination. According to studies, these mutations were in the spike that enable it to escape from antibodies brought up by either a prior coronavirus infection or immunization [82]. Omicrons’ new subvariant BA.2, highly contagious, has been discovered. The BA.2 was the most contagious but the least lethal of all the variants that were in existence. BA.2 variant has vaccine resistance, and the majority of illnesses in the fifth wave were breakthrough infections [83].

Corona Sixth Wave in Pakistan

Duration: June 22 to present: On June 22, 2022, a new rapidly – moving Omicron sub-Variant was discovered in Pakistan, particularly in Karachi, and is currently being transmitted locally. Health experts have issued a warning that the increase in new infections could become the sixth wave of the pandemic based on recent changes in the spike of Omicron variant cases around the nation. According to the figures, Karachi has been the province’s most impacted city with 340 confirmed cases. A 10.69% positive rate was registered in the port city within seven days, as reported by the health authority. Agha Khan University reported that Hospitalization has grown in the sixth wave, although not as significantly as it happened in the Delta (4th) wave. However, some people who exhibited severe symptoms were admitted to hospitals [37]. Nine deaths and 872 cases were reported in Pakistan on July 8, 2022. Additionally, the national positive rate reached over 4% for one week before falling to 3.77 percent as the number of tests administered rose by 30 percent. This wave experienced a much lower Active cases &

death rate and a much higher recovery rate than the previous waves as shown in Figure 2 & Figure 3. Contrary to earlier waves, which were stopped by effective lockdowns and tight commitment to SOPs in public places, there were no such preventive measures in place nationwide. Because of over crowdedness, transportation hubs like train and bus stations turned into the hotspot for super-spreader outbreaks.

According to data from the National Command and Operation Centre (NCOC), nine persons died because of this variant. More information from the data revealed that the number of daily Covid tests—which had previously been at just over 15,000—had jumped to 23,125. Due to more testing, the positivity rate in Pakistan remained below 7 percent, with Karachi topping the list with 6.49 percent. Although the NCOC had not formally announced the 6th wave of the pandemic, it was clear that cases were increasing both in Pakistan and globally. The lifting of travel restrictions abroad was the cause. The sub-variant BA.5 of Omicron is currently circulating throughout the world, but the good news is that it only causes bronchitis and does not progress to pneumonia. Additionally, there are very few illnesses and deaths that aren’t just virus-related but are caused by comorbidity. Lung bronchitis is an illness that irritates and inflames the lungs’ major airways. Comorbidity seems to be the simultaneous presence of multiple diseases, such as diabetes, high blood pressure, and cardiac issues [84].

Two novel Omicron subvariants (B.4 and B.5) have emerged in the sixth wave because of negligence from SOPs. Resuming the practice of socially isolating yourself in busy public spaces whilst wearing face masks should be advised. Otherwise, the advent of these two new Omicron subvariants could put the nation in a bad situation. The National Institute of Health in Islamabad has  used genome sequencing to identify these new sub-variants. These two new sub-variants, which are causing almost all the infections countrywide, are rapidly spreading throughout the nation.

Due to successive genetic variations, new variants of SARS-CoV-2 emerged. Table 1 summarizes the variants experienced or observed in all six waves along with their origin and earliest date. The present Omicron variant has the most diverged genetic variations. Other factors contributing to its fast spreading include the fact that existing COVID vaccines lose their effectiveness 6 months after the second dose, the public’s resistance to receiving a booster dose, and the general public’s lack of preventative behavior [85]. During October 2022 BA.5 and its descendent lineages became dominant around the globe [13].